Activin is a protein growth and differentiation factor with effects on a diverse set of biological processes, including reproduction, neural function, erythropoiesis, and embryonic development. The long term objectives of this proposal are to characterize the full complement of cell surface receptors for activin, and to analyze the intracellular signaling mechanisms by which binding to those receptors stimulates cells. At least two high affinity activin binding proteins, the type I and type II receptors, have been identified by affinity labeling activin-responsive cells. The recent elucidation of the structure of two closely related type II activin receptors (ActRII, ActRIIB) revealed these molecules to be prototypic members of a new family of ligand-activated protein serine/threonine kinases, suggesting a novel mechanism for activin-induced cellular signaling. The two aims of this project are 1) to characterize in detail the enzymatic properties of the ActRIIs and their relationship with the type I receptor, and 2) to identify putative target molecules that could serve as substrates for the ActRII kinases and to clarify their roles in the activin signaling pathway. Dimerization, both with other type II receptors and with the type I receptor will be studied by co- immunoprecipitation experiments; the importance of dimerization for receptor function will be analyzed by measurement of kinase activity. The regulation of kinase activity by ligand binding and by phosphorylation of ActRII itself will be studied. Sites of phosphorylation will be mapped and their importance for receptor function assessed. The identification of target molecules will be pursued by three means: by a genetic cloning strategy in yeast designed to isolate interacting proteins, by direct cloning using labeled kinase domain to probe cDNA expression libraries, and by purification of kinase-associated proteins. Target molecules will be assessed for their capacity to serve as substrates for the ActRII kinases both in vitro and in vivo, and for their functional involvement in the activin signaling pathway. By furthering our understanding of the basic mechanisms through which activin promotes cellular changes, the information generated by these experiments will provide insight into the molecular events which regulate activin responses. Because the substrate molecules identified by this project will likely serve as mediators for related ligands, the clarification of the activin signaling pathways will have a significant impact on the study of many fundamental biological systems.